Download Proinflammatory profile of in vitro monocytes in the ageing is

Pinke et al. Immunity & Ageing 2013, 10:22
http://www.immunityageing.com/content/10/1/22
IMMUNITY & AGEING
RESEARCH
Open Access
Proinflammatory profile of in vitro monocytes in
the ageing is affected by lymphocytes presence
Karen Henriette Pinke1, Bruno Calzavara1, Patricia Freitas Faria1, Magda Paula Pereira do Nascimento1,
James Venturini2 and Vanessa Soares Lara1*
Abstract
Background: Aging is associated with complex and constant remodeling of the immune function, resulting in an
increasing susceptibility to infection and others diseases. The infections caused by Gram-negative microorganisms,
present in nursing homes and hospitals, constitute one of the most common infections in the elderly, and are mainly
combated by innate immune cells. Although the functions of innate immunity seem more preserved during aging
than of adaptive immune mechanisms, two systems operate in an integrated way in the body, so that injury in one
part of the immune system inevitably affects the other as they are part of a defensive network. The aim of this study
was to investigate the in vitro production of proinflammatory (TNF-α, IL-6, IL-1β, CXCL-8 and MCP-1) and antiinflammatory (TGF-β and IL-10) cytokines by monocytes, stimulated or not (basal) with lipopolysaccharide, from healthy
young and elderly subjects. By means of PBMCs, we also studied if cytokine profile is altered in these different patient
groups, in the presence of lymphocytes, under the same experimental conditions.
Results: The monocytes from elderly presented higher basal production of TNF-α, MCP-1 and lower of TGF-β than
young monocytes. PBMC showed similar cytokines production, irrespective age or stimulation presence. In the
presence of lymphocytes, the spontaneous production of IL-10 was higher and of TGF-β was lower than monocytes,
regardless of age. After LPS-stimulation, the presence of lymphocytes resulted in increased IL-6, IL-1β, MCP-1 and IL-10
and decreased CXCL-8 and TGF-β in comparison to pure culture of monocytes from young patients. With age, the
same differences were observed, except for CXCL-8 and TGF-β which production was the same between monocytes
and PBMC stimulated with LPS.
Conclusion: These findings reinforce the systemic state of inflamm-aging frequently reported in elderly and considered
a factor of susceptibility to numerous diseases. Still, the cytokine production from just monocytes of the elderly showed
alterations, while in the lymphocyte presence not, suggesting an immunomodulator role of lymphocytes on
monocytes. In addition, the differences between the production patterns by LPS-stimulated PBMC between young and
elderly volunteers can be related with an imbalance in response against Gram-negative bacteria in throughout life.
Keywords: Immunosenescence, Monocytes, PBMC, Cytokines, Inflamm-aging
Background
In recent decades, the number of elderly has increased
considerably in comparison with young people and there
is a trend towards continuing increase in the twenty-first
century [1]. With these changes in life expectancy of the
population, there has been increasing interest in studies
related to health and quality of life during aging. Advancing age affects various cellular and biological functions,
* Correspondence: [email protected]
1
Department of Stomatology, Bauru School of Dentistry, University of São
Paulo, Al. Dr. Octávio Pinheiro Brisola, 9-75, 17012-901, Bauru, SP, Brazil
Full list of author information is available at the end of the article
including immune cells [2-9], mostly related to changes
in the equilibrium between cell survival, proliferation
and death. In vitro experiments have shown that absence
of the enzyme telomerase leading to critical shortening
of the protective ends of chromosomes plays a central
role in human cell senescence [10,11]. These changes in
immunosenescence are related to various impairments
such as susceptibility to inflammatory, infectious and
autoimmune diseases, as well as increasing rates of
occurrence of malignancies [1,12-14]. Although these
diseases are usually easily resolved clinically, elderly humans
suffer from complications caused by the individual's altered
© 2013 Pinke et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Pinke et al. Immunity & Ageing 2013, 10:22
http://www.immunityageing.com/content/10/1/22
response [1]. Much of this deterioration of immunity in
elderly is related to predisposition to infectious diseases,
such as caused by Gram-negative microorganisms that are
frequent in nursing homes and hospital settings [15,16]
and mainly combated by innate immune cells. Although
the functions of innate immunity appear to be more preserved during aging than the mechanisms developed by
the adaptive immune system [8,17], recent evidence has
shown that most innate immune functions are at least
partly affected during aging [5-7,18-20]. Thus, changes related to immunosenescence could affect the defense
mechanism as a whole; however, this complex process is
not yet completely understood.
Mononuclear phagocytes cells compromise macrophages
and their monocyte precursors and linage-committed bone
marrow precursors. Both macrophages and peripheral
blood monocytes exhibit morphological heterogeneity and
functional plasticity [21]. Lipopolysaccharide (LPS), an
endotoxin present in the cell wall of Gram-negative bacteria and widely used for immunological assay, is recognized by monocyte/macrophage CD14 receptor and tolllike receptor (TLR) 4-MD2 [22]. These molecular interactions result in the release of different active molecules in
various inflammatory mechanisms including interleukin-1
(IL-1), prostaglandin E2 (PGE2) and tumor necrosis factor
alpha (TNF-α) [22,23], which may be further amplified by
lymphocytes, the main source of important cytokines such
as interferon-gamma (IFN-γ) [24-26]. Peripheral blood
mononuclear cells (PBMC) are composed by monocytes
and lymphocytes and represent an important line of
defense against infection [27-30]. Moreover, human PBMC
culture is a classical and well-known test for evaluation of
the immunological status and for mimicking changes that
occur in infectious lesions and tissue remodeling and
repair after inflammation [21].
During immunosenescence, the role of T lymphocytes is
modified and related mainly to changes in intracellular
signaling pathways [31], with decreased generation of
lymphoid precursors [1,32] and their ability to proliferate
[14]. With advancing age, changes in monocytes are
mainly related to decline in the production of cytokines
such as IL-1, and decrease in tumoricidal activity and
superoxide production [33]. Whereas, monocytes from
aged individuals are associated with increased levels of
circulating interleukin-6 (IL-6) and TNF-α [34], characterizing the state called inflamm-aging, i.e., chronic systemic
inflammation in the elderly [14]. This persistent proinflammatory profile results from high antigenic exposure
throughout life to various microorganisms, including bacteria, which can change macromolecules such as deoxyribonucleic acid (DNA) or proteins by oxidation, acylation
or glycosylation. Theses altered molecules can stimulate
the innate immune response, particularly macrophages
via TLRs [35].
Page 2 of 10
The better understanding of the changes in immunomodulatory interplay between lymphocytes and monocytes during aging and in the cooperation between the
cells in this defense process could elucidate the immune
mechanisms of the elderly associated with increased susceptibility to infectious diseases, especially those caused
by Gram-negative bacteria [1,14-16,36]. Therefore, this
study investigated the in vitro production of cytokines
TNF-α, IL-6, IL-1β, CXCL-8, MCP-1, TGF-β and IL-10
by PBMC and monocytes from elderly and young volunteers after LPS stimulation.
Results
Monocytes from elderly subjects spontaneously produced
more TNF-α, MCP-1 and less TGF-β than young. The
values of cytokine levels detected from monocytes or
PBMC obtained from young or elderly persons were demonstrated as median, minimum, maximum and 1th, 3th
quartiles (Tables 1 and 2). The main findings are schematized in Figures 1 and 2. The basal production of TNF-α
and MCP-1 by monocytes from elderly group was higher
than those derived from young individuals. However, the
basal TNF-α production by PBMC showed a fall in elderly,
becoming below than young, although without statistical
difference (Tables 1 and 2). On the other hand, the TGF-β
production without stimulus by monocytes from elderly
was lower than young, while this production was not
detected by PBMC from all subjects (Figure 3).
Although the basal production has been different in
some conditions between young and elderly subjects, LPSstimulated monocytes and PBMC from both age groups
produced similar cytokines levels (Figures 1, 3 and 4).
In PBMC, LPS increased more the cytokines production than in monocytes. In the most experimental conditions, the LPS caused increase of the cytokine levels in
relation to basal production, except MCP-1 and TGF-β
(Figures 3 and 4).
On the comparative analysis within age groups, in the
presence of lymphocytes, the spontaneous production of
IL-10 was higher and of TGF-β was lower than that of
monocytes, regardless of age. However, LPS-stimulated
PBMC from young or elderly produced more IL-1β, IL-6,
MCP-1 and IL-10 than its respective monocytes (Figures 2,
3 and 4). Besides, the presence of lymphocytes under the
LPS-stimulation resulted in lower production of CXCL-8
and TGF-β in comparison to pure culture of monocytes in
young but not in elderly (Figures 2, 3 and 4).
Discussion
According to the vitro results of this study addressing
cytokines, the monocytes suffer changes throughout life.
The spontaneous production of the TNF-α and MCP-1
by monocytes were increased in aged volunteers compared with those of young individuals. However, the
Pinke et al. Immunity & Ageing 2013, 10:22
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Table 1 Cytokines production by monocytes or PBMC from young subjects
Cytokines
Cellular type
Production type
Median
Minimum
Maximum
1th quartile
3th quartile
TNF-α
Monocyte
Basal
0.0
0.0
24.4
0.0
0.0
LPS
246.6
34.5
1486.5
182.8
97.6
PBMC
Basal
2.5
0.0
19.0
0.0
10.4
LPS
341.6
70.8
1165.8
270.3
728.7
Monocyte
Basal
2.6
0.0
68.1
0.0
8.9
LPS
175.8
50.5
282.0
138.3
186.1
PBMC
Basal
0.0
0.0
123.2
0.0
6.4
LPS
139.6
0.0
266.6
116.7
218.2
Monocyte
Basal
0.0
0.0
0.1
0.0
0.0
LPS
69.6
15.3
173.7
37.5
173.7
PBMC
Basal
0.0
0.0
1.0
0.0
0.0
LPS
17.9
0.0
113.8
0.0
31.5
Monocyte
Basal
173.1
48.7
288.8
130
240.1
LPS
279.5
177.3
297.7
252.3
290.9
PBMC
Basal
144.8
64.0
215.1
118.6
185.4
LPS
229.8
218.4
305.4
219.8
303.0
Monocyte
Basal
6.4
0.0
190.5
0.0
39.8
LPS
48.2
0.0
241.2
0.0
148.4
PBMC
Basal
25.9
5.9
412.4
12.2
97.5
LPS
85.0
0.0
415.3
29.1
172.7
Monocyte
Basal
621.2
621.1
621.4
621.1
621.3
LPS
621.2
621.0
621.6
621.1
621.2
PBMC
Basal
51.7
8.9
220.5
25.8
130.9
LPS
567.2
110.2
690.5
223.1
587.9
Monocyte
Basal
0.0
0.0
5.9
0.0
0.0
LPS
149.3
33.8
654.8
55.4
323.8
PBMC
Basal
0.0
0.0
11.8
0.0
0.0
LPS
129.1
47.0
274.1
102.7
195.6
IL-6
IL-1β
CXCL-8
MCP-1
TGF-β
IL-10
Values showed by median, minimum, maximum, 1th and 3th quartiles obtained by descriptive statistical analysis (Statsoft Software Inc. Tulsa, Ok, USA).
TGF-β basal production was lower with senescence.
These immune differences between young and aged
people may represent some aspects of the complex and
constant remodeling of the immune system with advancing age, and contribute with a proinflammatory profile.
Among the variations already found and consistent with
our results is a high TNF-α concentration detected in
the plasma from the elderly [2,37,38], which may be related to the development of acute and chronic inflammatory conditions, carcinogenesis, and autoimmune
diseases [1,12-14]. Irrespective of age, the TNF-α
dysregulation could be associated to several diseases,
such as diabetes type II, rheumatoid arthritis and atherosclerosis, among others [39]. Although this change in the
pro-inflammatory profile appears to represent an evolutionary programming to infections resist [40], we not
found a relationship between high levels of TNF-α and
increased protection against the Gram-negative microorganisms in elderly. MCP-1 is a chemokine that regulates
the monocyte and memory T cells migration to sites of
antigen-induced inflammation [27,41,42] and is produced
mainly in response to inflammatory stimulus [43-45]. Increased levels of MCP-1 in elderly have been correlated
with the preservation of the memory T cell population and
progression of atherosclerosis [46,47]. Our findings about
the increase on basal production of the MCP-1 in elderly
could represent a systemic alteration in the traffic of the
monocytes and the memory T cells with aging.
Unlike our findings, other studies showed higher levels
of TGF-β from macrophages with aging [48,49]. This
cytokine have a complex and pleiotropic activity, presenting anti-inflammatory action on various cell types,
like mast cells, T cells [50-53] and proinflammatory
function on monocytes, attracting to the local aggression
Pinke et al. Immunity & Ageing 2013, 10:22
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Table 2 Cytokines production by monocytes or PBMC from elderly subjects
Cytokines
Cellular type
Production type
Median
Minimum
Maximum
1th quartile
3th quartile
TNF-α
Monocyte
Basal
0.0
0.0
253.1
0.0
98.4
LPS
489.8
141.6
935.2
378.8
572.2
PBMC
Basal
0.0
0.0
98.4
0.0
0.0
LPS
556.1
336.3
1425.1
433.0
694.7
Monocyte
Basal
0.0
0.0
0.1
0.0
0.1
LPS
363.2
319.8
391.8
329.4
389.6
PBMC
Basal
0.0
0.0
0.1
0.0
0.1
LPS
321.1
246.4
489.4
283.2
405.8
Monocyte
Basal
0.3
0.0
6.8
0.0
3.7
LPS
332.5
276.1
362.3
293.3
358.3
PBMC
Basal
0.0
0.0
12.3
0.0
6.1
LPS
323.0
280.0
336.7
299.9
331.4
Monocyte
Basal
59.5
9.3
213.2
11.8
158.9
LPS
202.8
191.1
219.6
193.3
214.8
PBMC
Basal
160.1
16.7
197.5
82.3
184.9
LPS
206.5
200.6
224.5
200.7
218.4
Monocyte
Basal
4.6
0.0
125.1
0.0
125.1
LPS
999.6
988.0
1039.2
988.0
1039.2
PBMC
Basal
314.8
85.2
362.3
85.2
362.3
LPS
994.4
948.2
1013.4
948.2
1013.4
Monocyte
Basal
0.0
0.0
0.1
0.0
0.1
LPS
23.3
0.0
78.6
0.0
62.6
PBMC
Basal
0.0
0.0
0.1
0.0
0.1
LPS
117.0
0.0
397.8
0.0
315.9
Monocyte
Basal
24.6
0.0
358.6
0.0
271.2
LPS
903.4
447.2
3115.4
701.3
1044.9
PBMC
Basal
28.1
0.0
508.2
12.2
52.4
LPS
966.8
490.3
1139.2
649.8
1031.2
IL-6
IL-1β
CXCL-8
MCP-1
TGF-β
IL-10
Values showed by median, minimum, maximum, 1th and 3th quartiles obtained by descriptive statistical analysis (Statsoft Software Inc. Tulsa, Ok, USA).
and inducing the release of IL-1 and IL-6 by these
cells [50,54].
Taken together, these results about spontaneous production of cytokines, obtained from elderly subjects corroborate the state of inflamm-aging reported in others
works [55-57], which appears to predispose elderly persons to diseases such as Alzheimer's, angina and osteoporosis [58-60]. Besides, it could alter the defense
mechanisms against microorganisms [57,61] and reduce
the ability to provide the immediate response to pathogens, as well as integrate and influence the acquired immune response [61].
Regarding PBMC, our analyzes showed no significant
difference between the basal production of cytokines
by elderly in relation to young. Already in stimulatedconditions, PBMC produced higher levels of IL-1β, IL-6,
MCP-1 and IL-10 than matched monocytes, irrespective
of age. However, CXCL-8 and TGF-β production from
stimulated PBMC was lower than matched monocytes
just in young group. Some trends involving these production patterns in elderly also can be highlighted. The
lymphocytes seem to increase the TNF-α and MCP-1
spontaneous production in the elderly, but not in young.
Besides, PBMC from young tend to produce more MCP1 and less CXCL-8, whereas in advance of age, these
cells seem to produce more TNF-α and TGF-β. Thereby,
our dates indicate interplay between the two cell types
that may have altered the production of cytokines from
monocytes, suggesting an immunomodulador role of
lymphocytes on monocytes. Also, the modified production pattern after LPS stimulus in the presence of the
lymphocytes can indicate a different modulation in the
elderly than in the young, under infectious conditions
caused by Gram-negatives.
Pinke et al. Immunity & Ageing 2013, 10:22
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Figure 1 Main differences between the cytokines production by monocytes and PBMC from elderly subjects in relation to young. With
increasing age, monocytes showed higher basal production of TNF-α, MCP-1 and lower of TGF-β than monocytes from young.
Figure 2 Influence of lymphocytes presence on cytokines production. Note that with the presence of lymphocytes the spontaneous
production of IL-10 was higher and of TGF-β was lower than that of monocytes, regardless of age. After stimulation with LPS, the presence of
lymphocytes resulted in increased IL-6, IL-1β, MCP-1 and IL-10 and decreased CXCL-8 and TGF-β in comparison to pure culture of monocytes
from of young patients. With age, the same differences were observed, except for CXCL-8 and TGF-β which production was the same between
monocytes and PBMC stimulated with LPS.
Pinke et al. Immunity & Ageing 2013, 10:22
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Figure 3 Measurements of the anti-inflammatory cytokines production by blood monocytes (MON) or PBMC from healthy elderly and
young subjects. Peripheral blood was obtained from volunteers, and the cells were purified as described in the Material and Methods section.
Monocytes and PBMC were challenged with LPS (100 ng⁄mL), or not (Basal), for 18 and 24 hours, respectively, and cytokine production was
determined by ELISA. Columns represent the median and error bar the interquartile range. The results were evaluated by Mann–Whitney Rank
Sum Test. In the same subject group, statistical differences are represented by continuous lines (cells stimulated or not) and letters for different cell
types. Asterisks indicate statistical difference between groups of subjects (p values - *0,0016; a and b 0,0039; c 0,0159; d 0,0010, e, f and g <0.0001).
These changes may be related to changes already characterized in lymphocytes with aging. Among them is the
variation in lymphocyte population in PBMC. Whereas
PBMC from young subjects presents 40,6% of the naïve,
36,6% of the memory and 16,8% of the effector/cytotoxic
T cells, in elderly, the lymphocyte population more numerous is of the memory CD8+ T cells (54%) with great
reduction on naïve T cells population (3,6%), [62,63].
Thus, despite the phenomena of immunosenescence being
more related to the cells of the specific immunity system
[8,17], the findings of the present study may represent an
important change in innate immunity cells from the elderly, modulated by cells of the specific immunity system,
strengthening the hypothesis that the interaction between
cells is an important step affected during remodeling
of the immune system in advancing age. Studies on
immunosenescence have shown controversies, including a
decreased production of interleukin-12 (IL-12) by the
monocytes of elderly persons in comparison with those of
young persons, and similar production of IL-10 by monocytes from young or elderly persons [2,33,34,64-66]. The
results of the present study also revealed similar levels of
IL-10 produced by monocytes from young and elderly persons, unlike TNF-α. Higher IL-10 production by PBMC
compared with isolated monocytes was expected, since
lymphocytes are major producers of IL-10. Thus, we tend
to believe that the most important alterations observed in
the present study could be attributed to monocytes, particularly in the presence of interaction with lymphocytes.
Other alterations in T cell populations in the elderly
[67-69] cannot be ruled out. Further studies will be needed
for better clarification of the molecular mechanisms involved in interaction process between immune cells and
pathogens, in the elderly.
Conclusions
In summary, it was found that in vitro monocytes from
aged volunteers spontaneously produced more TNF-α,
MCP-1 and less TGF-β in comparison with those from
young persons, corroborating the state of inflamm-aging
capable of predisposing persons to numerous diseases.
However, in presence of lymphocyte the cytokines levels
were equal between age groups, even after LPS challenge,
suggesting an immunomodulador role of lymphocytes on
monocytes. Still, after LPS challenge, the pattern of cytokines production in presence of lymphocyte was different
between age groups, which may suggest an imbalance in
the response against Gram-negative bacteria. Thus, our
data may indicate that cooperation between lymphocytes
and monocytes from peripheral blood can decrease a basal
state of inflammation present the elderly. However in infectious conditions, this modulation can be altered and
lead to a lower monocyte recruitment, proliferation and
differentiation of other cell types in elderly.
Methods
Sample population
Elderly and young adults were recruited from the Bauru
School of Dentistry, University of São Paulo (USP), and
enrolled in the study. Immunocompromised subjects were
excluded, including individuals with human immunodeficiency virus infection (HIV), diabetes mellitus requiring
medication, and those taking immunomodulating medications. Subjects who were pregnant, smokers and those
under treatment with antipsychotic drugs were also excluded. This study was approved by the Ethics committee
at the USP (Protocol No.100/2008, 051/2008 and 172/
2009). Informed consent was obtained from all volunteers
in compliance with Resolution 196/96 of the National
Pinke et al. Immunity & Ageing 2013, 10:22
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Figure 4 Measurements of the proinflammatory cytokines production by blood monocytes (MON) or PBMC from healthy elderly and
young subjects. Peripheral blood was obtained from volunteers, and the cells were purified as described in the Material and Methods section.
Monocytes and PBMC were challenged with LPS (100 ng⁄mL), or not (Basal), for 18 and 24 hours, respectively, and cytokine production was
determined by ELISA. Columns represent the median and error bar the interquartile range. The results were evaluated by Mann–Whitney Rank
Sum Test. In the same subject group, statistical differences are represented by continuous lines (cells stimulated or not) and letters for different
cell types. Asterisks indicate statistical difference between groups of subjects (p values - *0,0487; a 0,0029; b 0,0068; c 0,0061; d 0,0195; e 0,0180;
**
0,0399; f 0,0089; g 0,0098).
Council of Health. The inclusion criteria comprised
healthy subjects in ranging from 20 to 50 years old
(young) and from 60 to 85 years old (elderly) [70].
with the Türk dye staining, and suspended in complete
medium at a concentration of 1×106 PBMC/mL.
Monocytes isolated
PBMC isolated
PBMC were obtained by centrifugation of heparinized
venous blood over Histopaque 1083 gradients (SigmaAldrich, St. Louis, MO, USA) (400 × g for 28 minutes).
The cell suspensions recovered at the interface were
washed and resuspended in complete medium (RPMI
1640 supplemented with 10% heat-inactivated fetal bovine
serum, 100 U/mL penicillin and 100 μg/mL streptomycin).
Cell viability, as determined by 0.2% trypan blue dye exclusion, was > 95% in all experiments. PBMC were counted
To obtain the monocyte (MON) suspension, PBMC
were isolated as previously described. The monocytes
were counted using neutral red (0.02%), and were
suspended in complete medium at a concentration of
1×106 monocytes/mL. After this, were distributed (1.0 mL/
well) in 24-well plates with a flat-bottomed coverslip in
each well to allow adherence of monocytes and immunofluorescence testing on the glass coverslips. After incubation for 2 h at 37°C in a humidified atmosphere of 5%
CO2, non-adherent cells were removed by aspiration and
Pinke et al. Immunity & Ageing 2013, 10:22
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each well was rinsed twice and resuspended in complete
medium. Cell viability was assessed by morphological
analysis of the cells on coverslips with marking using the
anti-CD14-FITC and labeling of the cell nucleus via
DAPi. Over 98% of viable cells were adherent monocytes
that had intensely expressed the CD14 receptor, and
were morphologically viable.
Cell challenge with lipopolysaccharide (LPS)
Monocytes and PBMC were incubated with (LPS) or
without (Basal) LPS of Escherichia coli O55B5 (SigmaAldrich, St. Louis, MO, USA) (100 ng/mL - Mon; 1 ng/
mL-PBMC) for 18 and 24 h, respectively, at 37°C in 5%
CO2. Culture supernatants were harvested and stored
at −80°C until assayed.
Quantification of cytokines
Cytokine concentrations were determined in cell-free supernatants obtained after 24 h PBMC cultures and 18h
monocyte cultures, with or without LPS. The method utilized was enzyme-linked immunosorbent assay (ELISA),
using BD OptEIA™ kits (BD Biosciences) for TNF-α, IL-6,
IL-1β, CXCL8, MCP-1, IL-10 and TGF-β. The evaluations were performed according to the manufacturer’s
instructions.
Statistical methods
All dates showed p values of less than 0.05 for the Normality
Test (Shapiro-Wilk) and were considered non-parametric.
For statistical analysis, was utilized the Mann–Whitney
Rank Sum Test with p values <0.05 considered significant.
The software Statistica 11.0 (Statsoft Software Inc. Tulsa,
Ok, USA) was used.
Competing interests
The authors have no financial conflict of interests.
Authors’ contributions
KHP carried out the immunoassays, assay development, data analyses,
statistical analyses, and drafted the manuscript. BC participated of the
immunoassays and assay development. PFF participated in the design of the
study and helped with editing of the manuscript. MPPN helped to
immunoassays, assay development and to draft the manuscript. JV helped in
the assay development and helped to draft the manuscript. VSL conceived
of the study, participated in your design and coordination and helped to
draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
The authors thank Professors José Roberto Pereira Lauris and Heitor Marques
Honório (Department of Pediatric Dentistry, Orthodontics and Public Health,
Bauru School of Dentistry, USP) for the statistical analysis. They also thank
Msc. Márcia Sirlene Zardin Graeff and Marcelo Milanda Ribeiro Lopes for their
technical support and Margery Galbraith for valuable English corrections.
Author details
1
Department of Stomatology, Bauru School of Dentistry, University of São
Paulo, Al. Dr. Octávio Pinheiro Brisola, 9-75, 17012-901, Bauru, SP, Brazil.
2
Laboratory of Experimental Immunology, Department of Biological Sciences,
Faculty of Science, São Paulo State University, Av Eng Luiz Edmundo C,
14-01, 17033-360, Bauru, SP, Brazil.
Page 8 of 10
Received: 14 September 2012 Accepted: 2 June 2013
Published: 8 June 2013
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doi:10.1186/1742-4933-10-22
Cite this article as: Pinke et al.: Proinflammatory profile of in vitro
monocytes in the ageing is affected by lymphocytes presence. Immunity
& Ageing 2013 10:22.
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